Project 1 Project Summary The overall goal of our research is to elucidate the cellular and molecular mechanisms that underly muscular dystrophies in order to facilitate the rational design of novel therapeutic strategies and quantitative assays to assess target engagement and the effectiveness of therapeutic approaches. Our current research focuses on the dystroglycanopathies, a group of congenital/limb-girdle muscular dystrophies caused by defects in post-translational processing of the extracellular matrix (ECM) receptor ?-dystroglycan (?-DG). ECM proteins that contain laminin-G-like (LG) domains bind to ?-DG via a unique heteropolysaccharide [-GlcA-?1,3-Xyl-?1,3-]n called matriglycan. Genetic studies have shown that mutations in any one of at least eighteen genes encoding enzymes required for ?-DG post-translational processing lead to the absence of or a reduction in matriglycan and thus impair ?-DG receptor function. Although significant progress has been made in the identification and characterization of dystroglycanopathy genes, we still do not fully understand the biochemical and physiological function of matriglycan, or how its absence or reduction in length causes muscular dystrophy. The overall objective of the proposed research is to provide mechanistic insights into the role matriglycan plays in ?-DG receptor function and in the pathophysiology of the dystroglycanopathies. The overarching hypothesis of our research is that a thorough understanding of (a) the shortened matriglycan structure and resulting ?-DG receptor dysfunction in dystroglycanopathy patients and (b) the mechanisms underlying the pathophysiology of the dystroglycanopathies, will lead to more reliable diagnostics and novel therapeutic strategies. Specific Aim 1 will establish the relationship between ?-DG matriglycan length and laminin-binding properties of matriglycan on ?-DG in control and dystroglycanopathy patient fibroblasts and muscle biopsies. These studies will reveal abnormalities in the post-translational processing of ?-DG that result in shorter matriglycan with reduced affinity for laminin leading to muscular dystrophy. Specific Aim 2 will define the biochemical regulation of matriglycan synthesis and its role in the receptor function of ?-DG. These studies will define the requirements for matriglycan synthesis and how its synthesis is regulated by protein-protein and protein-sugar interactions. Specific Aim 3 will define novel mechanisms underlying the pathophysiology of the dystroglycanopathies and determine the structure and laminin binding properties required to improve muscle function. These studies will use myd mice with established dystrophic muscle pathology that are evaluated before and after LARGE gene transfer. Collectively, these aims will provide an understanding of the pathological mechanisms underlying dystroglycanopathies, which will be needed to develop a rationale for the design of novel diagnostic and therapeutic strategies, as well as approaches to monitoring therapeutic engagement and efficacy.